Analysis of the vitamin D family of metabolites, including, vitamin D3, vitamin D2, and metabolites of vitamin D3 and vitamin D2, such as the 25-hydroxy and the 1,25 dihydroxy analogs, has conventionally been difficult because these analytes are present at low levels in a determining matrix such as plasma. Conventional methods of analyzing vitamin D3, vitamin D2, and their metabolites suffer additional drawbacks. Immunoassays and LC/UV methods used to analyze vitamin D3 and its metabolites, for example, are not specific and suffer from sample treatment complexity. Also strategies involving LC/MSMS for exploiting derivatization of the analytes have failed to achieve the limit of detection required for the assay to be viable in a clinical setup and also lack multiplexing capability (Shimada, et.al., Analyst, December 1991, Vol 116, 1393-1397; Higashi et al., Chem. Pharm. Bull., 54(11), 1479-1485 (2006)). A need exists for a method of quantitating these analytes that overcomes these drawbacks.
It has been found that two functionalities in vitamin D3, vitamin D2, and metabolites of vitamin D3 and vitamin D2, which can be exploited for derivatization, include the hydroxyl group and the conjugated diene functionality. The Cookson reagent described by Shimada, et al. and Higashi et al. takes advantage of Diels-Alder chemistry to add a triazolone derivative across the diene functionality in vitamin D3, vitamin D2, and their metabolites.